Neurofilaments (NFs) are neuron-specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine- serine-proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo, selectively in the axonal compartment of neurons. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axon caliber and conduction velocity. Recent studies from our laboratory have shown that the mitogen-activated protein kinases (MAP kinases, or extracellular signal regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF-M and NF-H tail domains in vitro. However, it is not clear whether activation of the MAP kinase pathway can phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF-M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor (EGF), which induced the MAP kinase cascade in NIH 3T3 cells, also activated endogenous ERk1 and Erk2 and NF-M tail domain phosphorylation in the transfected cells. These results present direct evidence that in vivo activation of Erk1 and Erk2 is sufficient for NF-M tail domain phosphorylation in transfected cells. We have also demonstrated that activation of endogenous Erk1/2 by membrane depolarization and calcium influx through L-type calcium channels resulted in phosphorylation of the NF-tail domain in PC12 cells. This phosphorylation was inhibited in the presence of nifedipine, an L-type calcium channel blocker, and PD98059, a specific MEK1 inhibitor. These studies suggest a mechanism linking MAP kinase signal transduction to phosphorylation of neurofilaments. These findings provide significant new insights into mechanisms involved in neurofilament phosphorylation. - neurofilaments, phosphorylation, MAP kinases, topographic,regulation,phosphatases